This proposal is focused on understanding the role of the pipecolate-incorporating enzyme RapP in the rapamycin biosynthetic pathway. Rapamycin, a macrocyclic natural product with potent immunosuppressive properties, is synthesized by a combination of polyketide synthases (PKSs) and RapP, a nonribosomal peptide synthetase (NRPS). These enzymes have generated much attention because their modular nature may provide the opportunity for directed combinatorial syntheses. In particular, hybrid PKS/NRPS systems are of special interest as they could be utilized to increase the diversity of such syntheses. RapP is a 1542 amino acid, four domain, 160 kDa NRPS that adds the amino acid pipecolate to the completed polyketide chain, then cyclizes the chain to form the macrolactone. With the goal of understanding the function of each of the four domains of RapP, the specific aims of this proposal are to: (A) characterize the activity of the adenylation and peptidyl carrier protein domains using radioactivity incorporation assays, (B) demonstrate the formation of a peptide bond between acyl substrates and the pipecolyl-enzyme intermediate (C1 domain function), and (C) elucidate the determinants of macrolactonization (C2 domain function) using a variety of synthetically prepared substrates.